The present invention relates to the field of immunology and, in particular, to a vaccination procedure for protection of a host against disease caused by infection with a bacterium of the Chlamydiacease genus, particularly Chlamydia trachomatis. 
Chlamydia trachomatis is a species of the genus Chlamydiacease, order Chlamydiales, C. trachomatis infects the epithelia of the conjunctivae and the genital tract, causing trachoma and a variety of sexually transmitted diseases (STDs) which can lead to, respectively, blindness or infertility. There are at least 15 serovars of C. trachomatis, of which A, B and C are causative agents of trachoma, while serovars D, E, F, G, H, I, J and K are the most common causative agents of the Chlamydial STDs. C. trachomatis infections are endemic throughout the world. Trachoma is the leading cause of preventable blindness in developing nations, and it is estimated that 600 million people suffer from trachoma worldwide, with as many as 10 million of them being blinded by the disease. In the United States, there are an estimated 3 million cases per year of STDs caused by C. trachomatis. 
The pathogenesis of trachoma involves repeated ocular infections and the generation of a deleterious hersensitivity response to chlamydial antigen(s) (refs. 1 to 4xe2x80x94Throughout this specification, various references are referred to in parenthesis to more fully describe the state of the art of which this invention pertains. Full bibliographic information for each citation is found at the end of the specification). The available evidence supports the hypotheses that both secretory IgA and cell-mediated immune responses are important components of protection. Ocular infection in a primate model induces rapid and persistent production of IgA in tears, whereas the presence of IgG in tears is transient, corresponding to the period of peak conjunctival inflammation (refs. 5). Protective immunity following experimental ocular infection in a sub-human primate model is homotypic and resistance to ocular challenge correlates with the presence of serovar-specific antibodies in tears (refs. 1, 2, 6). Tears from infected humans neutralized the infectivity of homologous but not heterologous trachoma serovars for owl monkeys eyes (ref. 7) whereas passive humoral immunization with antitrachoma antibodies was not protective (ref. 8). Several lines of evidence indicate the importance of cell-mediated responses in protection from or clearance of chlamydial infection. B-cell deficient mice can resolve infection, whereas nude mice become persistently infected. Adoptive transfer of at least some chlamydia-specific T-cell lines or clones can cure persistently infected nude mice, and this anti-chlamydial activity is probably a function of the ability of the T-cells to secrete interferon-xcex3(refs. 9 to 16)
Past attempts to develop whole-cell vaccines against trachoma have actually potentiated disease by sensitizing vaccinees (refs. 1, 2). Sensitization has been determined to be elicited to a 57 kD stress response protein (SRP)(HSP60) present in all serovars of C. trachomatis. Repeated exposure to the 57 kD SRP can result in a delayed hypersensitivity reaction, causing the chronic inflammation commonly associated with Chlamydial infections. Thus, an immunogenic preparation capable of inducing a strong and enduring mucosal neutralizing antibody response and a strong cellular immune response without sensitizing the host would be useful (ref. 17).
A most promising candidate antigen for the development of a vaccine is the chlamydial major outer membrane protein (OMP) (refs. 18 to 20). Other surface proteins and the surface lipopolysaccharide are also immunogenic, but the antibodies they induce have not been found to be protective (refs. 21, 33). The MOMP, which is the predominant surface protein, is an integral membrane protein with a mass of about 40 kDa which, with the exception of four variable domains (VDs) designated I, II, III and IV, is highly conserved amongst serovars. The sequences of all four VDs have been determined for fifteen serovars (refs. 23, 24). Antibodies capable of neutralizing chlamydial infectivity recognize the MOMP (refs. 25, 26, 27, 28). Epitopes to which MOMP-specific neutralizing monoclonal antibodies bind have been mapped for several serovars (refs. 21, 22, 29, 30, 31, 32, 33), and represent important targets for the development of synthetic or subunit vaccines. The binding sites are contiguous sequences of six to eight amino acids located within VDs I or II, and IV, depending on the serovar. Subunit immunogens (e.g. isolated MOMP or synthetic peptides) containing MOMP epitopes can induce antibodies capable of recognizing intact chlamydiae (ref. 25). However, conventionally administered subunit immunogens are generally poor inducers of mucosal immunity. It would be useful to formulate chlamydial antigens in such a way as to enhance their immunogenicity and to elicit both humoral and cell-mediated immune responses.
Immune stimulating complexes (ISCOMs) are cage-like structures formed from a mixture of saponins (or saponin derivatives), cholesterol and unsaturated fatty acids. The components of ISCOMs are held together by hydrophobic interactions, and consequently proteins which are naturally hydrophobic (such as MOMP) or which have been treated to expose or add hydrophobic residues can be efficiently incorporated into the ISCOMs as they form (refs. 34, 35, 36).
C. trachomatis naturally infects the mucosal surfaces of the eye and genital tract. Local antibody and local cellular immune responses are an important component of protection from mucosal infections. Consequently, it would be useful for a chlamydial vaccine to induce a mucosal immune response including both cellular and antibody components.
DNA immunization is an approach for generating protective immunity against infectious diseases (ref. 37). Unlike protein or peptide based subunit vaccines, DNA immunization provides protective immunity through expression of foreign proteins by host cells, thus allowing the presentation of antigen to the immune system in a manner more analogous to that which occurs during infection with viruses or intracellular pathogens (ref. 38). Although considerable interest has been generated by this technique, successful immunity has been most consistently induced by DNA immunization for viral diseases (ref. 39). Results have been more variable with non-viral pathogens which may reflect differences in the nature of the pathogens, in the immunizing antigens chosen, and in the routes of immunization (ref. 40). Further development of DNA vaccination will depend on elucidating the underlying immunological mechanisms and broadening its application to other infectious diseases for which existing strategies of vaccine development have failed.
The use of attenuated bacteria, in particular S. typhimurium, has recently been reported for delivery of plasmid DNA for genetic immunization (refs. 41, 42). This type of delivery offers the added benefit of delivering the DNA to cell types that induce a specific immune response, such as a mucosal immune response. This type of vaccination also offers the advantages of being safe, as many safe, attenuated strains of Salmonella are readily available, and cost effective.
EP 0192033 B1 and U.S. Pat. No. 5,770,714 describe the provision of a DNA construct for the expression, in vitro, of Chlamydia trachomatis MOMP polypeptides comprising the following operably linked elements:
a transcriptional promoter,
a DNA molecule encoding a C. trachomatis MOMP polypeptide comprising a MOMP polynucleotide at least 27 base pairs in length from a sequence provided in Appendix A thereto, and
a transcriptional terminator, wherein at least one of the transcriptional regulatory elements is not derived from Chlamydia trachomatis. There is no disclosure or suggestion in this prior art to effect DNA immunization with any such constructs.
Copending U.S. patent application Ser. No. 08/893,381 filed Jul. 11, 1996 now U.S. Pat. No. 6,235,290 (WO 98/02546), assigned to University of Manitoba and the disclosure of which United States Patent Application is incorporated herein by reference, describes an immunogenic composition for in vivo administration to a host for the generation in the host of a protective immune response to a major outer membrane protein (MOMP) of a strain of Chiamydia, comprising a non-replicating vector comprising a nucleotide sequence encoding a MOMP or MOMP fragment at generates a MOMP specific immune response, and a promoter sequence operatively coupled to the nucleotide sequence for expression of the MOMP or MOMP fragment in the host; and a pharmaceutically-acceptable carrier therefor.
Copending U.S. patent application Ser. No. 08/713,236 filed Sep. 16, 1996 now U.S. Pat. No. 6,464,979 (WO 98/10789), assigned to Connaught Laboratories Limited and the disclosure of which United States Patent Application is incorporated herein by refer nce, describes an immunogenic composition, comprising the major outer membrane protein (MOMP) of a strain of Chlamydia, which may be Chlamydia trachomatis, and an immunostimulating complex (ISCOM).
The present invention provides a novel immunization strategy to provide protection against disease caused by infection of members of the Chlamydiae family, particularly Chlamydia trachomatis and materials used therein. The immunization strategy provided herein leads to a stronger protective immune response than other strategies.
According to one aspect of the invention, there is provided a method of immunizing a host against disease caused by infection by Chlamydia which comprises:
initially administering to the host an immunoeffective amount of an attenuated bacteria harbouring a nucleic acid sequence encoding at least one immunoprotective-inducing Chlamydia protein or fragment thereof which generates a Chlamydia protein-specific immune response, operatively connected to a eukaryotic expression element, such as the cytomegalovirus promoter, and
subsequently administering to the host an immunoeffective amount of at least one purified Chlamydia protein or fragment thereof which generates a Chlamydia protein specific immune response, of the same at least one Chlamydia protein or immunogenic fragment thereof as used in the initial administration, to achieve a Chlamydia specific protective immune response in the host.
The attenuated bacteria may be an attenuated strain of Salmonella or Shigella and the nucleic acid sequence may be the MOMP gene or fragments thereof from a strain of Chlamydia, including Chlamydia trachomatis and Chlamydia pneumoniae. The boosting protein can be the MOMP protein or immunogenic fragments thereof from a strain of Chlamydia, including Chlamydia trachomatis and Chlamydia pneumoniae. 
The administration steps may be effected to mucosal surfaces, such as by intranasal administration or by an initial intranasal administration of DNA followed by intramuscular administration of Chlamydia protein.
The immune response which is achieved in the host by the method of the invention preferably includes the production of Chlamydia-specific protection against live Chlamydia challenge and enhanced immunogenicity with greater delayed-type hypersensitivity (DTH) responses and high IgG2 and IgG1 antibody responses than achieved in other immunization procedures.
In another aspect, the present invention includes an attenuated strain of a bacterium harbouring a nucleic acid molecule encoding at least one immunoprotection-inducing Chlamydia protein or a fragment thereof which generates a Chlamydia protein specific immune response. The bacterium preferably is a strain of Salmonella, such as a strain of Salmonella typhimurium. The invention extends to such attenuated strain of a bacterium when used as an immunogen and to the use of such attenuated strain in the manufacture of an immunogen for administration to a host.
The present invention, in a further aspect, provides a method of immunizing a host against infection caused by a strain of Chlamydia, which comprises:
administering to the host an immunoeffective amount of an attenuated bacteria harbouring a nucleic acid molecule encoding at least one immunoprotection-inducing Chlamydia protein or a fragment thereof which generates a Chlamydia protein specific immune response. Any of the embodiments described herein with respect to the priming administration in the prime-boost immunization protocol described herein applies to this aspect of the invention.